The chlamydial inclusion remains largely uncharacterized as to its cellular origins. Neither fluid phase markers nor markers for various stages of the endocytic/lysosomal pathway are associated with the chlamydial inclusion. The mature inclusion therefore appears disconnected from endocytic pathways. However, fluorescent sphingomyelin endogenously synthesized from a Golgi-specific lipid, C6-NBD-ceramide, is translocated from the Golgi apparatus to Chlamydia trachomatis inclusions in a time, temperature, and energy dependent manner where it is rapidly incorporated into the cell walls of the intravacuolar chlamydiae. Approximately one-half of the newly synthesized sphingomyelin in C. trachomatis infected cells is trafficked unidirectionally to the inclusion. The transport of sphingomyelin to the inclusion is direct and specific to the chlamydial inclusion. NBD- sphingomyelin, incorporated into the plasma membrane, is not delivered to the chlamydial inclusion. Other Golgi-specific markers including various lectins and resident protein markers of the trans-Golgi demonstrate the close association of the Golgi apparatus with the chlamydial inclusion although none of these markers appear to be incorporated into the inclusion membrane. A novel experimental approach has identified secreted chlamydial proteins that are inserted into the inclusion membrane. The first of what appears to be multiple chlamydia- specified proteins in the inclusion membrane has been cloned, sequenced, and expressed. Based upon these observations, we suggest that the chlamydial inclusion represents an abberent vesicle derived from the trans-Golgi network, modified by the insertion of chlamydial proteins, and that inclusion membrane growth may be supported by the interuption of a cellular exocytic pathway.